CH615222A5 - Process for the preparation of a new naphthacene derivative - Google Patents

Abstract

The substance of the formula below is obtained by reduction of carminomycin by the microbiological route. This substance possesses antitumour activity at doses ranging from 0.125 to 0.250 mg/kg i.p. in mice. It is active against leukaemia L 1210, leukaemia P 388 and leucosarcomatosis. <IMAGE>

Description

The present invention relates to a process for the preparation of a new naphthacene derivative of formula:

0 011

CllOH-CH.

(I)

HO 0

and its salts.

The product of formula (I), which will be designated by the number 32999RP, is of particular interest due to its antitumor activity.

32999RP is a basic, dark red compound that forms salts with acids.

The 32999RP hydrochloride is soluble at a rate of approximately 100 mg / cm3 in methanol, pyridine, dimethylformamide and water, approximately 10 mg / cm3 in n-butanol saturated with water at 20 ° C, approximately 5 mg / cm3 in ethanol, and practically insoluble (less than 0.1 mg / cm3) in acetone, chloroform, methylene chloride, ethyl acetate, benzene, methyl isobutyl ketone, dioxane and hexane.

The 32999RP hydrochloride contains carbon, hydrogen, oxygen, nitrogen and chlorine. Its elementary composition is close to:

C 56.6 H 5.7 O 29.41 N 2.39 Cl 6.0%.

CH,

It is further characterized by the following physicochemical properties:

Appearance: microcrystalline powder, orange-red.

Melting point: around 200 ° C (with decomposition).

Rotatory power: (determined in 0.09% solution in ethanol

0.1% HCl IN).

[a] g0 = + 208 ° ± 32 °.

Ultraviolet spectrum: determination from a solution of 10.65 mg / 1 in ethanol with 0.1% IN HCl.

Maximum absorption at

233 nm 254 nm 292 nm

Ej%

I cm

620 495 140

This spectrum is represented by FIG. 1.

Visible spectrum: determination from a solution of 10.65 mg / 1 in ethanol with 0.1% IN HCl.

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Maximum absorption at

493 nm 512 nm 527 nm p i% 1 cm

272

189

190

This spectrum is represented by FIG. 2.

Infrared spectrum (determination from tablets mixed with KBr):

This spectrum is represented by FIG. 3 on which we have plotted on the abscissa, on the one hand, the wavelengths expressed in microns (upper scale) and, on the other hand, the wave numbers in cm-1 (lower scale) and on the ordinates the optical densities.

Table 1 shows the main infrared absorption bands for this product expressed in number of waves (cm-1):

3410 F

1930 th.

1370 th.

985 F

750 m

485

m

3250 th.

1900 tf

1315 th.

960 th.

725 m

475

th.

3220 th.

1800 tf

1285 F

930 m

708 m

465

f

3050 th.

1735 th.

1255 m

915 th.

695 th.

450

th.

2970 F

1690 th.

1235 F

885 m

665 th.

438

m

2930 F

1635 th.

1195 F

875 m

640 th.

415

tf

2900 th.

1600 tF

1165 F

845 th.

625 th.

390

m

2860 th.

1580 th.

1115 F

840 th.

600 m

320

f

2820 th.

1510 th.

1062 F

820 F

580 f

2680 th.

1460 F

1050 th.

810 th.

565 f

2600 th.

1440 F

1030 th.

780 m

540 f

1980 tf

1405 F

1010 F

762 m

530 f

tF = very strong F =

: strong m

= medium f = low

tf = very low thickness. = shoulder.

32999RP can be characterized by thin layer chromatography on silica gel using as the developing solvent the methylene chloride / formic acid / methanol mixture (80/17/3 by volume) at a temperature of 24 ° C. In this system , the 32999RP hydrochloride has an Rf close to 0.2.

The acid hydrolysis of 32999RP or one of its salts makes it possible to obtain the product of formula:

GHOH-CH,

Maximum absorption at

234 nm 254 nm 293 nm

F i% I cm

770 640 190

i% 1 cm

This spectrum is represented by FIG. 4 on which curves I and II correspond respectively to solutions at 50 and 5 mg / 1. Visible spectrum: determined from a 10 mg / 1 solution in ethanol containing 0.1% IN HCl.

5 Maximum absorption at

493 nm 370

515 nm 255

528 nm 270

10

This spectrum is represented by FIG. 5.

Infrared spectrum (determination from tablets mixed with KBr):

This spectrum is represented by FIG. 6 on which we have plotted on the abscissa on the one hand, the wavelengths expressed in microns (upper scale) and, on the other hand, the wave numbers expressed in cm-1 (lower scale) and in ordered the optical densities.

Table 2 shows the main infrared absorption bands for this product expressed in number of waves (cm-1).

25

30

3500 th.

1705

f

1255 tF

1005 m

785 th.

570 f

3420 F

1640

th.

1240 th.

975 m

775 th.

545 f

3360 th.

1600

tF

1195 F

950 m

765 m

530 tf

3080 th.

1580

th.

1165 F

925 m

740 m

505 th.

2975 m

1540

th.

1130 m

910 m

710 m

485 m

2925 m

1.458

F

1095 f

885 m

700 th.

465 m

2910 th.

1448

F

1070 th.

875 m

680 tf

450 m

2890 th.

1412

F

1062 F

865 f

665 tf

435 m

2860 th.

• 1370

F

1050 th.

840 m

625 th.

385 tf

2680 th.

1315

th.

1030 m

815 F

595 f

360 tf

1980 tf

1285

tF

1020 m

805 th.

585 f

320 tf tF = very strong

F =

strong m =

: medium f = low

35

(II)

tf = very low thickness. = shoulder

The aglycone of 32999RP can be characterized by thin layer chromatography on silica gel using as the developing solvent the methylene chloride / formic acid / methanol mixture (80/17/3 by volume) at a temperature of 24 ° C. In this system, the aglycone has an Rf close to 0.7.

According to the present invention, 32,999 RP is obtained by microbiological reduction of the antibiotic called carminomycin which corresponds to the formula:

0 OH

COCH,

(III)

HO O OH OH

which is the aglycone of 32999RP.

32999RP aglycone is soluble at 20 mg / cm3 in pyridine, 5 mg / cm3 in methanol, dioxane, chloroform and methylene chloride and less than 0.1 mg / cm3 in acetone , ethyl acetate, hexane and water.

This product also has the following physicochemical properties:

Appearance: orange-red microcrystalline powder.

Melting point: around 185 ° C (with decomposition).

Elementary analysis: it is close to:

C 61.57 H 5.31 0 30.16%.

Rotatory power: (determined in 0.1% solution in dioxane).

["] D ° = + 249 ° + 32 °.

Ultraviolet spectrum: determination from solutions of 50 and 5 mg / I in ethanol with 0.1% IN HCl.

(VìYf wyyi

-OH

HO 0 OH 0-CH -

CH - CH - CH 2 I I NH „OH

- CH - CH,

Carminomycin and its preparation by culture of Actinomadura carminata Sp. Nov. Were described in French patent application No. 74.00349 which was published under number 2235700. The structure of carminomycin was published by MG Brazhnikova et al. , "J. of Antibiotics ”, XXVII, N ° 4, 254-259 (1974).

The microbiological reduction of carminomycin is generally carried out in an aqueous medium using either cultures of microorganisms which have reached a suitable stage of development, or cells isolated from these cultures, or enzymatic extracts obtained from these microorganisms.

The microorganisms which can be used according to the process of the present invention can belong to the category of streptomycetes or bacteria. Among the microorganisms which are particularly suitable, may be mentioned Streptomyces

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lavendülae (ATCC 8664), Streptomyces roseochromogenes (ATCC 13400), Corynebacterium simplex (ATCC 6946) or Bacterium cyclooxydans (ATCC 12673). The best results are obtained from Corynebacterium simplex (ATCC 6946).

The culture of the microorganism producing the enzymatic system capable of reducing carminomycin to 32999RP can be carried out by any method of aerobic culture at the surface or at depth, but the latter is to be preferred for reasons of convenience. The seeding and fermentation techniques and the various types of apparatus which are in common use in the fermentation industry are used for this purpose.

The culture medium of the microorganism must essentially contain sources of assimilable carbon and nitrogen, mineral elements and growth factors, all of these elements being able to be provided in the form of well-defined products or by complex mixtures such as they are found in organic products of various origins.

As sources of assimilable carbon, carbohydrates such as glucose or other carbonaceous substances such as sugar alcohols or certain organic acids can be used. Certain animal or vegetable oils, such as bacon oil or soybean oil, can advantageously replace or be added to these carbon sources.

The sources of assimilable nitrogen are extremely varied. They can be very simple chemical substances such as mineral or organic ammonium salts, urea, certain amino acids. They can also be provided by complex substances mainly containing nitrogen in protein form: casein, lactalbumin, gluten and their hydrolysates, soya flour, peanut, fish, peptone, extracts of meat, yeast, soluble distillers, corn-steep.

Among the added mineral elements, some can have a buffering or neutralizing effect such as alkaline or alkaline earth phosphates. Others provide the ionic balance necessary for the development of the microorganism, such as chlorides and sulfates of alkali or alkaline earth metals.

Growth factors are products of a vitamin nature, such as riboflavin, folic acid, pantothenic acid.

The pH of the fermentation medium at the start of the culture must be between 6 and 7.8 and preferably between 6.5 and 7.5. The optimum temperature for culturing the microorganism is 29-31 ° C, but satisfactory development is obtained at temperatures between 26 and 37 ° C.

The aeration of the culture medium can vary between fairly wide values. However, it has been found that aerations of 0.3 to 3 liters of air per liter of broth per minute are particularly suitable.

In addition, to obtain a culture having a good reducing activity, it is preferable to agitate the culture medium, for example by means of an agitator whose speed of rotation can vary between 100 and 250 rpm.

Generally, the culture of the microorganism reaches a satisfactory degree of development after a period of 24 to 48 hours.

The reducing activity essentially depends on the quantity of cells formed during the culture. With isolated cells, for example after centrifugation of the culture medium, the amount of 32999RP formed from carminomycin is directly related to the concentration of cells in the reaction medium; media rich in cells have a higher reducing power.

The reduction of carminomycin to 32999RP is carried out by bringing an aqueous solution of carminomycin or one of its salts into contact with the culture of the microorganism having reached a sufficient degree of development and reducing power, or with the isolated cells. of these cultures, or with enzymatic extracts obtained from these cells.

The enzymatic extracts of the cells can be obtained after destruction of the cell walls either by chemical or biochemical methods, or by physical methods. Preferably, the destruction of the cell walls is carried out from the culture of the microorganism or of the cells isolated from this culture in suspension in aqueous medium, or by treating them with an enzyme capable of lyzing the cell wall, such as lysozyme. , or by subjecting them to the action of ultrasound. The enzymatic extracts are in solution in the supernatant liquid which is obtained after centrifugation of the medium. Generally, the solution of the enzyme extracts is used without further treatment in the reduction phase.

Generally, the reduction is carried out in an agitated medium at a temperature between 23 and 37 ° C, preferably between 26 and 30 ° C and it is complete after 1 to 4 days of contact.

The reduction can take place at a pH between 5 and 10, but it is preferable to operate at a pH between 7 and 8.

The reaction medium can be stirred for example by a stirrer, the speed of rotation of which can vary between 100 and 250 rpm.

The reduction can be carried out either on carminomycin or one of its pure salts, or on an impure product, or on the acid filtrate of a culture which has produced carminomycin. To obtain good yields, the concentration of carminomycin in the reducing medium is advantageously between 0.1 and 1 g / 1 at the start of the reduction.

32 999 RP can be separated from its production medium according to the usual methods for isolating antibiotics belonging to this family.

For example, 32,999 RP can be extracted from the reduction or fermentation medium at a pH close to 9 using an organic solvent such as chloroform, methylene chloride, n-butanol or a mixture of these solvents in suitable proportions.

The fermentation must can also be filtered in the presence of a filter aid at a pH between 1.5 and 9. It is advantageous to carry out this operation in an acid medium, and in particular by acidifying at a pH between 1, 5 and 2 using oxalic acid. It is also possible to carry out filtration at a pH of between 2 and 8 in the presence of an aliphatic alcohol containing 1 to 3 carbon atoms. 32999RP can be extracted from the filtrate, after concentration, using an organic solvent such as chloroform, methylene chloride, n-butanol or a mixture of these solvents in suitable proportions.

32 999 RP can be isolated from organic extracts, after successive washing and extraction, by precipitation after concentration of the extracts to a small volume or by addition in a miscible solvent in which 32999RP is practically insoluble, such as hexane, after transformation optional antibiotic into an addition salt with an acid such as the hydrochloride.

The 32999 RP can optionally be purified by various conventional purification operations such as crystallization or chromatography on various adsorbents.

32999RP and its nontoxic, i.e. pharmaceutically acceptable, salts have been shown to be particularly active on graftable tumors of mice at doses between 0.125 and 0.250 mg / kg i.p. on L1210 leukemia, P388 leukemia and leukosarcomatosis.

The toxicity of 32999RP hydrochloride has been studied mainly in mice. Its 50% lethal dose (LD50) has been determined (treatment for 4 consecutive days) intraperitoneally and is close to 0.4 mg / kg.

The following examples, given without limitation, show how the invention can be put into practice. In the following:

a) the identification of 32999RP is carried out by thin layer chromatography on silica gel by comparing the Rf of the products obtained with that of carminomycin or of 32999RP in the same solvent system;

b) the production of 32999 RP is determined from chroma-tograms on a thin layer, that is to say from the compared intensities of the corresponding spots of the extract and that of

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32999RP pure taken as a reference, either by comparison of the peak areas recorded with the Chromo-scan plate reader, or finally by colorimetrically dosing the 32999RP eluted from the chromatographic support at the spot of the desired product.

Example 1

A culture medium A is prepared, the composition of which in g / 1 is as follows:

Brewer's yeast autolysate powder 15

Monopotassium phosphate 2.5

Dipotassium phosphate 2.5

Glucose anhydrous 15

A solution of the first three constituents in 1000 cm3 of distilled water is adjusted to pH 6.9 by addition of normal sodium hydroxide and is distributed in vials of 300 cm3 at the rate of 50 cm3 per vial. These vials are sterilized at 122 ° C for 20 min, then cooled. The medium is completed by sterile adding to each flask 1.5 cm3 of a sterile glucose solution at 500 g / 1. The pH of the medium is then 7.05.

80 of these flasks are seeded by adding 2.5 cm3 of an inoculum culture of the Corynebacterium simplex strain (ATCC 6946) to each flask. The culture is developed on a table stirred at 220 rpm in a chamber at 30 ° C. After 48 h, the culture is well developed and its pH is 7.70. An aqueous solution at 10 mg / cm 3 of carminomycin hydrochloride is then added at a rate of 2.5 cm 3 per vial. The reduction phase is continued on a table stirred at 220 rpm in an enclosure at 26 ° C.

After 48 h, the reduction is complete and the culture pH is 8.5. The contents of the 80 vials are combined. 50 cm3 of the culture are taken homogeneously, which is treated with 25 cm3 of a borate buffer so as to bring the pH to 9.0. Then extracted with 2 times 50 cm3 of a methylene chloride / normal butanol mixture (70/30 by volume). The organic extracts are dried over anhydrous sodium sulfate.

The production of 32999RP is determined by chromatography of the organic extracts on a thin layer of Silicagel. The volumes of extracts deposited are from 10 to 20 ni. The plates are developed with the following mixture: methylene chloride / formic acid / methanol (80/17/3 by volume) in order to obtain a good separation of carminomycin and 32999RP. The Rf of the products obtained are compared with those of the reference products chromatographed under the same conditions.

The quantitative evaluation of the production of 32999RP shows that the culture of Corynebacterium simplex (ATCC 6946) reduces carminomycin to 32999RP with an 80% yield on the carminomycin used.

Example 2

A culture medium A prepared as in Example 1 is prepared and distributed in 300 cm 3 flasks.

Culture medium B, the composition of which is as follows, is also prepared and distributed in 300 cm 3 flasks:

Peptone 10 g

Meat extract 5 g

Glucose 10 g

Distilled water q.s.p 1000 cm3

The pH is adjusted to 7.2 by addition of normal sodium hydroxide; the medium is sterilized at 122 ° C for 20 min.

A vial containing 50 cm3 of medium A whose pH is 7.05 and a vial containing 50 cm3 of medium B whose pH is 6.5 is seeded with, per vial, 2.5 cm3 of an inoculum culture of the Corynebacterium simplex strain (ATCC 6946).

The cultures are grown on a table stirred at 220 rpm in an enclosure at 30 ° C. After an appropriate culture time (48 h for medium A and 72 h for medium B), an aqueous solution

to 10 mg / cm3 of carminomycin hydrochloride is then added at a rate of 2.5 cm3 per flask to obtain a concentration of 0.5 g / 1 of carminomycin.

After these additions, cultures A and B are incubated for 2 and 4 days, respectively, on a table stirred at 220 rpm in an enclosure at 26 ° C. The flasks are treated under the conditions described in Example 1.

The yields in 32999RP, relative to the carminomycin involved, are as follows:

Medium Yield (%)

At 80

B 60

Example 3

Cultures on medium A of the strain Corinebacterium simplex (ATCC 6946) are prepared in 300 cm 3 flasks under the conditions described in Example 1. The cultures are grown on a table stirred at 220 rpm in an enclosure at 30 ° C.

After 30 h of incubation, 5 flasks are combined (250 cm3 of wort) and the cells present are harvested by centrifugation. The entire cell harvest is then resuspended in a 300 cm3 flask containing 50 cm3 of M / 15 phosphate buffer at pH 8.0. Another cell suspension is also prepared in the same way, using for the recovery of the cells 50 cm 3 of an M / 15 phosphate buffer at pH 5.0. Then added to each flask 2.5 cm3 of an aqueous solution at 4 mg / cm3 of carminomycin hydrochloride to obtain a concentration of 0.2 g / 1 of carminomycin.

The flasks are incubated for 48 h under the conditions described in Example 1 and are treated as in Example 1.

The yields of antibiotic 32999RP compared to the carminomycin involved are as follows:

pH Yield (%)

8.0 90

5.0 60

Example 4

A culture medium A, the pH of which is 6.8 and which is prepared as described in Example 1, is prepared and distributed in 300 cm3 flasks, at a rate of 50 cm3 per flask.

Several vials are seeded with a pH of 6.8 by adding 2.5 cm3 of an incoculum culture of the Bacillus cyclooxydans strain (ATCC 12673) to each. The culture is developed for 48 h on a table stirred at 220 rpm in an enclosure at 30 ° C.

The reduction of carminomycin is carried out using:

- either the entire culture whose pH is 7.65;

- Or a cell suspension in a pH 8 buffer prepared under the conditions described in Example 3.

1 cm3 of a 10 mg / cm3 aqueous solution of carminomycin is added to each flask to obtain a concentration of 0.2 g / 1.

The reduction phase is continued for 4 days on a table stirred at 220 rpm in an enclosure at 26 ° C. The flasks are treated under the conditions described in Example 1. The yields of 32999RP relative to the carminomycin used at stake are:

Reducing medium Yield (%)

Whole culture 94 Isolated cells 75

Example 5

Cultures on medium A of the Bacillus Cyclooxydans strain (ATCC 12673) are prepared under the conditions described in Example 4. To the whole cultures incubated for 48 h and whose pH is 7.65, carminomycin is added in the form:

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a) of an aqueous solution at 10 mg / cm 3 of the pure product, at a rate of 1 cm 3 per vial (0.2 g / 1 of carminomycin);

b) an aqueous solution at 10 mg / cm3 of a crude product which contains 16% carminomycin, at a rate of 4 cm3 per vial (0.13 g / 1 of carminomycin).

The flasks are then incubated for 4 days under the conditions described in Example 1 and treated in the same way. The yields in 32999RP, compared to the carminomycin involved are as follows:

Product Yield (%)

Pure carminomycin 94

Crude carminomycin 92

Example 6

A culture medium C is prepared, the composition of which in g / 1 is as follows:

Soy flour 30

Soybean oil (d = 0.92) 4.6

Monopotassium phosphate 1

Calcium carbonate 2.5

Glucose anhydrous 50

The constituents of the medium, with the exception of glucose, are dissolved or suspended in 900 cm3 of distilled water. The preparation, adjusted to a pH of 7.20 by addition of sodium hydroxide N, is distributed in vials of 300 cm3 at the rate of 45 cm3 per vial. These vials are sterilized at 122 ° C for 20 min and cooled. The medium is made up by sterile adding to each flask 5 cm3 of a sterile glucose solution at 500 g / 1. The pH of the medium ready for sowing is 7.

A 300 cm3 flask containing 50 cm3 of the above medium whose pH is 7 is inoculated with 2.5 cm3 of an inoculum culture of the Streptomyces roseochromogenes strain (ATCC 13400). The culture is developed for 48 h on a table stirred at 220 rpm in an enclosure at 30 ° C. The cells produced by this culture are then harvested. The reduction of carminomycin to antibiotic 32999RP is carried out using a suspension of the cells in 50 cm3 of an M / 15 phosphate buffer at pH 8, containing 1 cm3 of an aqueous solution at 10 mg / cm3 of carminomycin hydrochloride to obtain a concentration of 0.2 g / 1. The reduction phase is continued for 48 h on a table stirred at 220 rpm in an enclosure at 26 ° C. The cell suspension is treated under the conditions described in Example 1. The yield of 32999RP relative to carminomycin stake is 57%.

Example 7

A culture medium C prepared under the conditions described in Example 6 is prepared and distributed in 300 cm 3 flasks.

Vials containing 50 cm3 of this medium, whose pH is 6.90, are inoculated with 2.5 cm3 of an inoculum culture of the Streptomyces lavendulae strain (ATCC 8664). The culture is developed for 40 h on a table stirred at 220 rpm in an enclosure at 26 ° C. Then added to each flask containing the whole culture, the pH of which is 7.50.1 cm 3 of an aqueous solution at 10 mg / cm3 of carminomycin hydrochloride to obtain a concentration of 0.2 g / 1. The reduction phase is continued for 4 days under the same conditions of agitation and temperature. The vials are treated under the conditions described in Example 1. The yield of 32999RP relative to the carminomycin involved is 61%.

Example 8:

A. Fermentation

We load in a 1701 fermenter

Peptone 600 g

Meat extract 600 g

Yeast extract 600 g

Glucose monohydrate 600 g

City water q.s.p 1101

After adjusting the pH to 6.8 with 40 cm3 of 10N sodium hydroxide, the following is added:

Calcium carbonate 300 g

The pH of the medium is then close to 6.8; the medium is sterilized by bubbling steam at 122 ° C for 40 min; after cooling, due to the condensation of steam during sterilization, the broth volume is 1201 and the pH of 8.3. The pH is adjusted to 7 by adding 90 cm3 of 12N hydrochloric acid.

Inoculum is inoculated with 250 cm3 of an inoculum culture in a stirred io erlenmeyer flask of the Corinebacterium simplex strain.

(ATCC 6946). The culture is developed at 30 ° C for 32 h while shaking and aerating with sterile air; it is then suitable for sowing the productive culture.

The producing culture is carried out in an i5 8001 fermenter loaded with the following substances:

Brewer's yeast autolysate powder 6 kg

Monopotassium phosphate 1 kg

Dipotassium phosphate 1 kg

City water q.s.p 3551

After adjusting the pH to 7 by adding 460 cm 3 of 10N sodium hydroxide, the medium is sterilized by bubbling steam at 122 ° C for 40 min. After cooling, due to the condensation of steam during sterilization, the volume of the broth is 3851; it is completed at 4001 by addition of 151 of a sterile aqueous solution containing:

Glucose monohydrate 6 kg

The pH of the medium is 6.8. The inoculum culture is inoculated with 401 in the 1701 fermenter described above. The culture is developed at 30 ° C. by shaking with a turbine rotating at 160 rpm and aerating with a volume of sterile air of 20 m3 / h. After 30 h, the culture is suitable for carrying out the biochemical transformation of carminomycin into 32999RP.

Is introduced into the culture 41 of an aqueous solution containing:

35 Carminomycin hydrochloride 40 g

Incubation is continued for 17 h at 26 ° C, shaking and aerating under the conditions described above. Carminomycin is reduced to 32999RP with a yield close to 100%.

40 B. Extraction

To 4201 of wort obtained as described in Example 8A, 421 of n-butanol and 2.9 1 of 6N sodium hydroxide are added. The active principle is extracted against the current with n-butanol (60% by volume) on two centrifuges. The extract, whose volume is 3301, is concentrated under reduced pressure (5 to 10 mm of mercury) at 40 ° C to a volume of 201.

To the concentrated extract, 201 of methylene chloride is added and this mixture is washed with 101 of water brought to pH 10 by addition of sodium hydroxide. The washing water, which contains 32999RP, is extracted 2 times 50 by 101 from the methylene chloride / n-butanol mixture (80/20 by volume).

The three organic extracts are combined and then concentrated under reduced pressure (5 to 10 mm of mercury) to a volume of 151. 55 Then 0.21 of the n-butanol / 1 IN hydrochloric acid mixture (90/10) is added. by volume), then concentrated under reduced pressure (5 to 10 mm of mercury) to a volume of 51.

The antibiotic 32999RP in the form of the hydrochloride is precipitated by addition of the concentrate in 351 of hexane. The precipitate is drained, washed and dried. 52 g of crude hydrochloride of 32999RP are thus obtained.

C. Purification a) To a suspension of 50 g of crude hydrochloride of 32999RP, 65 obtained as indicated in Example 8B, in 130 cm3 of water, 520 cm3 of dioxane are added. The solution obtained is filtered.

The 32999 RP hydrochloride is crystallized by adding 41 dioxane, at a temperature in the region of 20 ° C, to the filtrate.

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The crystals are drained, washed with dioxane and dried. 27 g of semi-purified 32999RP hydrochloride are thus obtained.

b) 4.2 g of crude hydrochloride obtained as in Example 8B are dissolved in 0.21 of water. The pH is adjusted to 4.5 by addition of ION sodium hydroxide.

The antibiotic contained in this solution is fixed on a column containing 0.41 of Amberlite IRC 50 in acid form. The column is washed with 11 of water, 21 of 50% aqueous methanol, 21 of 90% aqueous methanol and finally with 31 of 90% aqueous methanol containing 1% of sodium chloride. The eluate is concentrated under reduced pressure (5 to 10 mm of mercury) at 40 ° C to a volume of 0.51. The concentrate, the pH of which is adjusted to 10 by addition of 1 IN sodium hydroxide solution, is extracted with twice 0.51 of chloroform and twice 0.51 of the chloroform / n-butanol mixture (80/20 by volume). The combined organic extracts are concentrated under reduced pressure (5 to 10 mm of mercury) at 40 ° C. During the concentration, 10 cm 3 of n-butanol containing 1% (by volume) of 11N hydrochloric acid and 5% ( by volume) of water. The concentrate, the volume of which is brought to 30 cm3, is added slowly to 500 cm3 of hexane. The precipitate obtained is drained, washed and dried. 1.26 g of semi-purified 32999RP hydrochloride are thus obtained.

The semi-purified product (27 g) obtained according to a) is dissolved in 300 cm3 of water. The solution obtained is filtered.

The 32999RP hydrochloride is crystallized by adding 2.71 acetone to the filtrate, at a temperature in the region of 20 ° C. The crystals are drained, washed and dried. 17 g of pure 32999RP hydrochloride are thus obtained.

Example 9

A culture of Corynebacterium simplex (ATCC 6946) grown for 30 h under the conditions described in Example 8A is sterile centrifuged at a temperature in the region of 0 ° C 50 cm3. After centrifugation, the cells are resuspended in 50 cm3 of M / 15 phosphate buffer at pH 7.5. The cells are then subjected to the action of ultrasound (25 kHz) for 15 minutes at a temperature close to 0 ° C. After centrifugation, 5 mg of carminomycin hydrochloride are added to the supernatant. The solution thus obtained is incubated under the conditions described in Example 1. After 40 h, the carminomycin is reduced to 32999RP with a yield close to 60%.

Example 10

A culture of Corynebacterium simplex (ATCC 6946) grown for 30 h under the conditions which are described in Example 8A is sterile centrifuged at a temperature in the region of 0 ° C. After centrifugation, the cells are resuspended in 50 cm3 of tris (hydroxymethyl) amino-methane / HCl M / 5 buffer at pH 8.1 and transferred to a 300 cm3 Erlenmeyer flask; 25 mg of EDTA (ethylenediaminetetraacetic acid), 25 mg of CTAB (cetyl-trimethylammonium bromide) and 200 mg of lysozyme are then added, then the flask is placed at 37 ° C. for 1 h on a rotary table at 220 t / min. After centrifugation, 5 mg of carminomycin hydrochloride are added to the supernatant. The solution thus obtained is incubated under the conditions which are described in Example 1. After 40 h, the carminomycin is reduced to 32999RP with a yield close to 25%.

Example 11

0.5 g of pure 32999RP hydrochloride are hydrolyzed by dissolving in 50 cm3 of water supplemented with 10 cm3 of 11N hydrochloric acid and heating to boiling for 2 h. A crystalline precipitate of aglycone forms during hydrolysis. The precipitate is drained, washed and dried. 0.3 g of pure aglycone is thus obtained.

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4 sheets of drawings

Claims (2)

615,222 CLAIMS 1. Process for the preparation of a new basic antitumor substance, designated by the number 32999 RP, the formula of which is 0 oh çhoh-cu3 -OH I. 1 OH Ô - CH - CH. -'CH - CH - CH - CH, 2 I I 3 NH2 OH in the form of a free base or of an addition salt with an acid, characterized in that the carminomycin of formula is reduced 0 OH CO-CH „ -OH > H Ô - GH - CH „- CH - CH 2 1 i NH. OH - CH - CH, or one of its salts, by means of a microorganism capable of reducing carminomycin or by means of enzymes originating from such a microorganism. 2. Method according to claim 1, characterized in that the chosen from Streptomyces lavendulae (ATCC 8664), Streptomyces roseochromogenes (ATCC 13400), Corynebacterium simplex (ATCC 6946) or Bacterium cyclooxydans (ATCC 12673), or cells isolated from this culture or reduction enzyme extracts is carried out by means of a culture of a microorganism 35 obtained from cells of these microorganisms.